This invention generally relates to electroosmotic surfaces exposed to buffers, and in particular to capillaries or channels having modified electroosmotic surfaces that are used for electrophoretic transport or separations, which permit the fall control of electroosmosis by an applied external voltage field.
Electroosmosis is the flow of liquid that is in contact with a solid, under the influence of an applied electric field, The movement of the fluid typically results from the formation of an electric double layer at the solid/liquid interface, i.e., the separation of charge that exists in a thin layer of the surface and in a thin layer of the fluid adjacent to the surface.
Typically electroosmostic flow is observed in capillary electrophoresis which employs a capillary tube having a silica inner surface and which utilizes one or more buffer fluids. In such a configuration electroosmosis arises from interaction of the electric double layer, which is present on the inner-surface/buffer interface of a silica tube, with the longitudinal voltage gradient, wherein the electroosmotic flow rate (xcexdeof) is defined by the following relationship:
xe2x80x83xcexdeof=xcex6(xcex5b/xcex7)Eapp=xcexceofxc2x7Eappxe2x80x83xe2x80x83(1)
where xcex6 is the potential drop across the diffuse layer of the electric double layer (commonly referred to as the xcex6 (zeta)-potential), xcex5b is the permittivity of the buffer solution, xcex7 is the viscosity of the buffer solution, xcexceof electroosmotic mobility, and Eapp is the voltage gradient across the length of the capillary or channel. The external flow control effect is directly related to the xcex6-potential through the changes in the surface charge density of the channel. The total surface charge density results from the chemical ionization ("sgr"si) and the charge induced by the radial voltage field ("sgr"rv), as described in Hayes et al., xe2x80x9cElectroosmotic Flow Control and Monitoring with an Applied Radial Voltage for Capillary Zone Electrophoresis,xe2x80x9d Anal. Chem., 64:512-516 (1992), which is incorporated herein by reference. According to the capacity model, the "sgr"rv is described by the following equation:
"sgr"rv=(xcex5QVr/ri)(1/ln(rori)xe2x80x83xe2x80x83(2)
where xcex5Q is the permittivity of the fused silica capillary, Vr is the applied radial voltage, ri is the inner radius of the capillary, and ro is the outer radius of the capillary. For a flat plate capacitor model the relationship is:
"sgr"rv=(xcex5QVrAe)/dxe2x80x83xe2x80x83(3)
where Ae is the projected area of the radial electrodes on the channel wall and d is the wall thickness in the flat plate capacitor. The surface charge density is related to the xcex6-potential by the following equation, as described in Bard, et al., Electrochemical Methods Fundamentals and Applications. Wiley and Sons (New York, 1980); Davies, et al., Interfacial Phenomena, 2nd Ed., Academic Press (New York, 1963); and Overbeek, Colloid Science, Kruyt ed., Vol. I, p. 194 (Elsevier, Amsterdam, 1952), which are incorporated herein by reference:
xcex6=exp(xe2x88x92xcexax)Eapp(xcex5b/xcex7)(2kT/ze)xc2x7sin hxe2x88x921[("sgr"si+"sgr"rv)/(8kTxcex5bn0)xc2xd]xe2x80x83xe2x80x83(4)
where
xcexa=(2n0z2e2/xcex5bkT)xc2xdxe2x80x83xe2x80x83(5)
and n0 is the number concentration, z is the electronic charge, e is the elementary charge, T is the temperature, xcexa is the inverse Debye length, x is the thickness of the counterion, and k is the Boltzmann constant.
Areas of the capillary, which are not under direct control of the external voltage, are still effected by the radial field by a mechanism attributed to surface conductance effects, as described in Wu, et al., xe2x80x9cLeakage current consideration of capillary electrophoresis under electroosmotic controlxe2x80x9d J. Chromatogr., 652:277-281 (1993); Hayes, et al., xe2x80x9cElectroosmotic Flow Control and Surface Conductance in Capillary Zone Electrophoresis,xe2x80x9d Anal. Chem., 65:2010-2013 (1993); and Wu, et al., xe2x80x9cDispersion studies of capillary electrophoresis with direct control of electroosmosis,xe2x80x9d Anal. Chem., 65:568-571 (1993). The magnitude of this effect may be approximated by a xcex6-potential averaging approach. The xcex6-potential in the uncovered zones is the average of the xcex6-potential in the controlled zones and the xcex6-potential from charge generated from the fused silica surface chemical equilibrium. The xcex6-potential for the surface chemical equilibrium may be obtained directly from flow measurements in the capillary without an applied external voltage, as described in Overbeek, at p. 194. The resulting flow (xcexdobs) through the capillary which is generated from these sections according to the following relationship, as described in Hayes et al., at pp.512-516:
xe2x80x83xcexdobs=xxe2x80x2xcexdr+(1xe2x88x92xxe2x80x2)xcexdavxe2x80x83xe2x80x83(6)
where xxe2x80x2 is the fraction of the capillary under the influence of the applied radial voltage (xxe2x80x2 greater than 0), xcexdr is the electroosmotic flow rate if the entire capillary were under radial voltage effects (which may be calculated from equations 1 and 4, with 2 or 3), and xcexdav is the average electroosmotic flow generated from surface charge due to chemical equilibrium and the surface charge in the controlled zone due to radial voltage effects.
The voltage gradient across the capillary also induces an additional movement of charged species according to:
xcexdem=(xcexceof+xcexcem)xc2x7Eappxe2x80x83xe2x80x83(7)
where xcexdem is the migration rate of a charged species, and xcexcem is the electrophoretic mobility of that charged species. Since xcexcem is constant under these experimental conditions, any change in xcexdem may be attributed to changes in xcexceof.
To obtain an expression directly relating changes in elution time (xcex94tel) and the change in surface charge density (xcex94"sgr"t), it is noted that elution time is tel=L/xcexdem, wherein L is the length of the capillary from the injector to the detector and xcexdem is the velocity of the analyte. The velocity of the analyte is described by equation 7 where the electrophoretic mobility of that charged species is a constant under these experimental conditions. Noting that xcexceo is equal to xcex6xc2x7(xcex5b/xcex7) (see equation 1) and the definition for tel, the following expression can be derived:
tel=L/[(xcex6(xcex5b/xcex7)+xcexcem)xc2x7Eapp].xe2x80x83xe2x80x83(8)
Equation 4 gives a function of xcex6 which includes a term for surface charge ("sgr"si+"sgr"rv) for both the chemically-generated surface charge and the external voltage-induced charge. For the surface coating assessments "sgr"rv=0 and "sgr"si is a function of the surface coating. It follows that upon coating the surface, the measured change in elution time can be used directly to calculate the change in the surface charge from the following equation:
xcex94tel=L/[({exp(xe2x88x92xcexax)xc2x7(2kT/ze)xc2x7sin hxe2x88x921[(xcex94"sgr"si)/(8kTxcex5bn0)xc2xd]xc2x7(xcex5b/xcex7)}+xcexcem)xc2x7Eapp]xe2x80x83xe2x80x83(9)
or by substituting A=exp(xe2x88x92xcexax)(xcex5b/xcex7)(2kT/ze) and B=1/(8kTxcex5bn0)xc2xd this simplifies to:
xcex94tel=L/[(Axc2x7sin hxe2x88x921[Bxcex94"sgr"si]+xcexcem)xc2x7Eapp].xe2x80x83xe2x80x83(10)
Noting that all variables in this expression except xcex94tel are constant under these experimental conditions and rearrangement results in a more useful form of this equality:
xcex94"sgr"si=[sin h{([L/(xcex94telEapp)]xe2x88x92xcexcem)/A}]/B.xe2x80x83xe2x80x83(11)
However, the usefulness of the external voltage technique is limited because it only provides control at low pH (e.g., less than pH 5) and low ionic strength buffers in standard systems.
External voltage to control fluid flow at higher buffer pH can be used if the surface charge generated by the chemical equilibrium at the buffer/wall interface is minimized, as described in Hayes, et al., xe2x80x9cEffects of Buffer pH on Electroosmotic Flow Control by an Applied Radial Voltage for Capillary Zone Electrophoresis,xe2x80x9d Anal. Chem., 65:27-31 (1993) and Poppe, et al., xe2x80x9cTheoretical Description of the Influence of External Radial Fields on the Electroosmotic Flow in Capillary Electrophoresis,xe2x80x9d Anal. Chem., 65:888-893 (1996), which are incorporated herein by reference. Minimization of the surface charge may be accomplished with surface coatings, such as coating including organosilanes, which can minimize analyte adsorption by silica surfaces for many separation techniques, including capillary electrophoresis, as described in Poppe, et al. at pp. 888-893 and Hjerten, et al., xe2x80x9cA new type of pH- and detergent stable coating for elimination of electroendoosmosis and adsorption in (capillary) electrophoresis,xe2x80x9d Electrophoresis, 14:390-395 (1993), which is incorporated herein by reference. Due to the labile silicon-oxygen-silicon-carbon bond (e.g., Sixe2x80x94Oxe2x80x94Sixe2x80x94C bond) between the silica surface and the organosilane, however, such organosilane treatments have been found to be unstable at either high or low buffer pH, as described in Hjerten, et al. at pp. 390-395; Kirkland, et al., xe2x80x9cSynthesis and characterization of highly stable bonded phases for high-performance liquid chromatography column packings,xe2x80x9d Anal. Chem., 61:2-11 (1989); and Vansant, et al., Characterization and Chemical Modification of the Silica Surface, (Elseiver, Amsterdam, 1995), which are incorporated herein by reference.
Application of coatings containing polymers to a capillary surface can also be used to eliminate the chemical equilibrium-based surface charge. As described in Srinivasan, et al., xe2x80x9cCross-linked polymer coatings for capillary electrophoresis and application to analysis of basic proteins, acidic proteins, and inorganic ions,xe2x80x9d Anal. Chem., 69:2798-2805 (1997), which is incorporated herein by reference, these coatings can minimize protein adsorption and eliminate or permanently change electroosmosis. Typically these polymers are covalently bound or physically adsorbed to the inner surface of the capillary, or used as dynamic coatings, i.e., buffer additives having surface-active properties so that the additives can adhere to the wall in an adsorbed/free-solution equilibrium. In addition to altering surface charge density, these polymers suppress electroosmosis by increasing viscosity within the electric double layer. Unfortunately, this local viscosity is unaffected by the potential gradients created by the external voltage fields, as described in St. Claire, xe2x80x9cCapillary Electrophoresis,xe2x80x9d Anal. Chem, 68:569R-586R (1996). The viscosity within the electric double layer significantly contributes to the frictional forces which retard movement of the entrained ions within the longitudinal voltage gradient, thereby directly impeding electroosmotic mobility. High-viscosity surface layers, therefore, produce low electroosmosis. In fact, high viscosity surface layers have been utilized to stop electroosmosis altogether, as described in Huang, et al., xe2x80x9cMechanistic Studies of Electroosmotic Control at the Capillary-Solution Interface,xe2x80x9d Analy. Chem., 65:2887-2893 (1993), which is incorporated herein by reference, and Srinivasan, et al., at pp. 2798-2805. Therefore, these polymer-coated approaches cannot be utilized in systems which require dynamic flow control by an applied radial field.
This deleterious increased viscosity effect can be minimized by monolayer surface coverage without the use of polymers or polymer-forming reactants. Capillaries coated with organosilane treatments to provide monolayer surface coverage have been reported, most notably for gas and liquid chromatography applications. These treatments have also been briefly explored for radial voltage flow control for capillary electrophoresis. One example is the use of commercially xe2x80x98deactivatedxe2x80x99 tubing to xe2x80x9c . . . yield[s] effective EOF [electroosmotic flow] control by applied radial voltage,xe2x80x9d as described in Hayes, et al., xe2x80x9cElectroosmotic Flow Control and Monitoring with an Applied Radial Voltage for Capillary Zone Electrophoresis,xe2x80x9d Anal. Chem., 64:512-516 (1992). Alternatively, a butylsilane monolayer surface has been used to improve the effectiveness of flow control, but resulted in a surface which was unstable above pH 5, as described in St. Claire, at pp 569R-586R; Huang et al. at pp. 2887-2893; and Towns, et al., xe2x80x9cPolyethyleneimine-bonded phases in the separation of proteins by capillary electrophoresis,xe2x80x9d J. Chromatogr., 516:69-78 (1990), which is incorporated herein by reference. While these coatings are specifically utilized for dynamic flow control, they are also unstable at pH extremes.
Electroosmosis can be used to move fluids through the small channels of instrumentation designed on single microchips, as well as in capillary electrophoresis. One limitation of using electroosmosis for fluid flow in both these applications is the lack of control and the poor reproducibility of the electroosmotic flow in standard commercial capillary electrophoresis systems.
Accordingly, there exists a need in the art for an inner-surface coating for the external voltage control of electroosmosis having several characteristics. First, the surface created must retain low surface charge density in the presence of the aqueous buffers typically used in capillary electrophoresis. Second, the surface charge density should be insensitive to pH changes of the buffer, thus remaining consistent over a large range of normally encountered pHs (e.g., 2-11) and buffer types. Finally, the surface created must not increase the viscosity of the solution near the surface.
Accordingly it is an object of the present invention to provide an arrangement and method for controlling electroosmotic flow of a fluid which can be used over a pH range of 2-11.
It is another object of the invention to provide an arrangement and method for controlling electroosmotic flow by maintaining low charge density at the electroosmotic surface.
A further object of the invention is to provide an arrangement and method for controlling electroosmotic flow which does not result in increased viscosity in surface layers near a fluid solid interface.
These objectives have been substantially satisfied and the shortcomings of the prior art have been substantially overcome by the present invention, which in one embodiment is directed to an electrophoresis apparatus including an electroosmotic surface comprising a substrate having hydroxyl groups and a coating on the substrate comprising a component formed by reacting a triorganosilane having a single leaving group with the substrate. In another embodiment, the electroosmotic surface comprises a silica and a substrate coating comprising a sterically hindered triorganosilane having a single leaving group which has reacted with the silica substrate.
In another embodiment, the present invention is directed to an electrophoresis apparatus including an electroosmotic surface comprising a substrate having surface hydroxyl groups; a coating on the substrate comprising an inert oxide; and a coating on the oxide surface comprising a component formed by reacting an organosilane having a single leaving group with the oxide surface.
In an additional embodiment, the present invention is directed to a process for providing an electrophoresis apparatus including an electroosmotic surface comprising a substrate having hydroxyl groups and a triorganosilane coating on the surface. The process includes the step of forming a coating on the substrate by reacting a triorganosilane having a single leaving group with the substrate.
In another embodiment, the present invention is directed to a process for providing an electrophoresis apparatus including an electroosmotic surface comprising a substrate having surface hydroxyl groups, an inert oxide coated on top of the substrate, and an organosilane coated on top of the oxide surface. The process includes the step of coating the substrate with an inert oxide and then forming a coating on the oxide surface by reacting the oxide surface with an organosilane having a single leaving group.